Endospore Staining Report

Endospore stain is the fourth staining method used to add contrast. “Bacteria capable of producing endospores do not do so uniformly during their culture’s growth. Sporulation is done in response to nutrient depletion” (Leboffe). The theory to stain endospores cells is that keratin in the spore resist the stain so a more extreme measure must be taken to stain the bacteria. Malachite Green, the primary stain, is forced into the spore by steaming the bacterial emulsion (Leboffe). Malachite Green is water soluble so spore mother cells can be decolorized, which leave the spore stained with a green-blue color and then the mother cell can be countered stained with Safranin. Besides the Malachite Green stain and the Safranin stain, a steam apparatus will be needed along with the bacteria Bacillus cereus and Bacillus subtilis. To start the process turn on the steaming

This is a differential stain in which the decolorization step occurs between the applications of two basic stains (Leboffe). The theory is that the decolorization is the most critical step in the process. Gram-negative cells are easily decolorized with alcohol or acetone whereas Gram-positive cells are not (Leboffe). Due to this, Gram-negative cells can take on the red colored, Safranin stain. The Gram-positive cells are already stained and cannot take up the Safranin. “The alcohol/acetone in the decolorizer extracts lipids, making Gram-negative wall more porous and incapable of retaining the crystal Violet iodine complex, thereby decolorizing it. Dehydration of the gram-positive wall by the decolorizer coupled with it's thicker peptidoglycan and greater degree of cross-linking trap the crystal Violet iodine complex more efficiently, making the gram-positive wall less susceptible to decolorization” (Leboffe). Materials needed are Crystal Violet stain, Gram iodine stain, decolorizer, and the bacteria Staphylococcus epidermidis and Escherichia coli in